Bradykinin type peptides

ABSTRACT

The substitution of the L-Pro at the 7-position with D-Phe or D-Tic and substitution of the L-Phe at the 8-position with hydroxyproline ethers and thioethers of the peptide hormone bradykinin and other additional substituted analogs of bradykinin converts bradykinin agonists into bradykinin antagonists. The invention further includes additional modifications at other positions within the novel 7- and 8-position modified bradykinin antagonists, which increase enzyme resistance, antagonist potency, and/or specificity of the new bradykinin antagonists. The analogs produced are useful in treating conditions and diseases of a mammal and human in which an excess of bradykinin or related kinins are produced or injected as by insect bites.

This application is a continuation of U.S. patent application Ser. No.07/866,385, filed Apr. 14, 1992, abandoned, the entire contents of whichare hereby incorporated, and which in turn is a continuation-in-part ofU.S. patent application Ser. No. 07/687,950, filed Apr. 19, 1991abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compounds which are bradykinin receptorantagonists, pharmaceutical compositions and methods for using thesecompounds to antagonize the effects of bradykinin in mammals, includinghumans. More particularly, the invention relates to the substitution ofthe L-Pro at position 7 with D-Phe or D-Tic and substitution of theL-Phe at position 8 with hydroxyproline ether or thioether compounds andits L-configuration intermediate product which convert bradykininagonists into antagonists and also includes additional modifications atother positions within the 7- and 8-position modified bradykininantagonist which confer increased antagonist potency, resistance toenzymatic degradation and/or tissue specificity on the D-aminoacid-containing bradykinin sequence.

2. Description of the Prior Art

Bradykinin (BK) is a nonapeptide generated as a result of the activityof kallikreins, a group of proteolytic enzymes present in most tissuesand body fluids, on kininogens. Once released, kinins produce manyphysiological responses, including pain and hyperanalgesia bystimulating C- and A-fibers in the periphery. There is also considerableevidence that kinins contribute to the inflammatory response.

Bradykinin, and its physiologically important related peptides kallidin(Lys-bradykinin) and Met-Lys-bradykinin, exhibit physiological actionswhich qualify them as mediators of inflammatory reactions, hypotensivestates, and pain. Bradykinin is overproduced in pathological conditionssuch as septic shock, anaphylaxis, rhinitis, asthma, inflammatory boweldisease, and certain other conditions including acute pancreatitis,post-gastrectomy dumping syndrome, carcinoid syndrome, migraine, andangioneurotic edema. The production of bradykinin from the plasmaresults in pain at the site of the pathological condition, and theoverproduction intensifies the pain directly or via bradykinin-inducedactivation of the arachidonic acid pathway which produces prostaglandinsand leukotrienes, the more distal and actual mediators of inflammation.

In addition to its analgesic and proinflammatory effects, bradykinin isa vasodilator. Because of its ability to lower blood pressure,bradykinin has been implicated in the pathogenesis of several shocksyndromes, particularly septic or endotoxic shock. Bradykinin is also apotent bronchoconstrictor in animals and asthmatic subjects and it hasbeen implicated as a contributor to the pathogenesis of airwayinflammatory conditions such as allergic asthma and rhinitis.

Thus a bradykinin inhibitor or bradykinin receptor antagonist isexpected to possess a number of desirable biological effects in thetreatment, for example, of inflammation, septic shock, asthma, burnpain, rhinitis, and allergy.

The search for understanding the mechanism of action of bradykinin,which is essential for the development of useful tools for diagnosticuse, and for the development of therapeutic agents aimed at alleviatingthe intense pain caused by the production and overproduction ofbradykinin, has been hindered by the lack of specific sequence-relatedcompetitive antagonists of bradykinin.

Several non-peptide, non-specific and non-selective antagonists of oneor more of the biological activities of bradykinin have been describedamong compounds as diverse as analgesics and anti-inflammatorysubstances, which act via the prostaglandin system and not directly onbradykinin biological receptors. These are antihistamines;bradykinin-antibodies; benzodiazepine derivatives; high molecular weightethylene oxide polymers; gallic acid esters; and serotonin inhibitors.None of these compounds or classes of compounds specifically inhibitbradykinin.

Heptyl esters of various amino acid-containing substances, such assingle basic amino acids, the dipeptide Phe-Gly and of analogs of C-terminal peptide fragments of bradykinin (i.e., Pro-Phe-Arg) have beenreported as anti-bradykinin substances. When tested in bradykinin assaysystems, they prove to be weak partial agonists/antagonists, dependingon the dose, with little specificity for inhibiting bradykinin action.

Preparations of damaged vascular tissue have been reported to respond tobradykinin analogs which lack the C-terminal arginine residue, but notto bradykinin itself, and analogs of these des-Arg(9)-bradykinins havebeen developed as antagonists for the non-physiological activity ofbradykinin. These antagonists have no significant bradykinin-likeagonist effects, nor any antagonist effect on any of the physiologicallysignificant kinin-responding systems. Furthermore, several bradykininanalogs containing the O-methyl ether of Tyr residues at positions 5and/or 8 have been reported to produce mixed agonist/antagonist activityon isolated uteri of galactosemic rats, but not on normal rats.

Other changes in the bradykinin molecule have been additions of aminoacids at the N-terminal end which affect the rate of enzymaticdegradation of bradykinin in vivo.

It has been reported that the half life of bradykinin in the systemiccirculation is less than 30 seconds. Bradykinin appears to be completelydestroyed (98-99% destruction) on a single passage through the pulmonarycirculation as determined in an anesthetized rat by measuring thedepressor effects of an agonist following intra-aortic (IA) (bypassingthe pulmonary circulation) and intravenous (IV) administration.Resistance of bradykinin agonists to pulmonary kininase destruction invivo also appears promoted by addition of single (i.e., D-Arg-, DLys-,Lys-) and double (DLys-Lys-) basic amino acid residues to the N-terminalof the bradykinin sequence. The addition of the dipeptide Lys-Lys to theN-terminal of bradykinin agonists has been reported to confer completeresistance to in vivo destruction on initial passage through thepulmonary circulation.

Several research groups have prepared bradykinin receptor antagonists.Stewart and Vavrek in U.S. Pat. No. 4,801,613, (which reference isincorporated in its entirety herein) disclose a series of bradykininantagonists wherein the L-Pro at the 7-position of the peptide hormonebradykinin or other substituted analogs of bradykinin is substitutedwith an aromatic amino acid of the D-configuration which convertsbradykinin agonists into bradykinin antagonists. The analogs producedare useful in treating conditions and diseases of a mammal and human inwhich an excess of bradykinin or related kinins are produced or injectedas by insect bites into the body. The specific L-Pro substitutions areselected from the group consisting of D-Nal, D-PNF, D-Phe, D-Tyr, D-Pal,D-OMT, D-Thi, D-Ala, D-Trp, D-His, D-Homo-Phe, D-Phe, pCl-D-Phe (CDF),D-Phg, D-Val, D-Ile, D-Leu, and MDY.

In U.S. Pat. No. 4,693,993, also to Stewart and Vavrek, additional L-Prosubstitution materials are disclosed.

Abandoned U.S. application Ser. No. 07/687,959, discloses and claimsadditional Bradykinin agonist materials.

U.S. Pat. No. 4,242,329 to Claeson et al. disclose the formation ofBradykinin-inhibiting tripeptide derivatives. A process for producingsaid tripeptide derivatives by synthesis and purification methods whichare known in the peptide chemistry is also disclosed as well aspharmaceutical preparations comprising the tripeptide derivative.

Published European Patent Application No. 0 413 277 A1 disclosesbradykinin antagonists as having natural or synthetic amino acidsincluding ring-constrained heterocyclic amino acids (e.g.,spiro(bicycl[2.2.1]heptan)-2,3-pyrrolidin-5-carboxylic acid) wherein thepeptides were prepared using standard solid phase FMOC technology. Thispublication also discloses that the G position, namely position 8, maybe a fragment of a heterocyclic ring system, whereby the preferredsubstituents on the heterocycles are: pyrrolidinyl-2-carboxylic acid,piperidinyl-2-carboxylic acid,1,2,3-4-tetrahydroisoquinolinyl-3-carboxylic acid,cis-and-trans-deca-hydroisoquinolinyl-4-carboxylic acid, cis-exo,trans-octahydroiso-quinolinyl-2-carboxylic acid, cis-endo, cis-exo,trans-octahydrocyclopenta-[b]-pyrrolyl-2-carboxylic acid orhydroxy-prolinyl-2-carboxylic acid.

SUMMARY OF THE INVENTION

The present invention resides in the discovery that the novel compoundsidentified below, are potent bradykinin receptor antagonists. Thecompounds are useful in the treatment of various diseases includinginflammatory disorders, asthma, septic shock and burn pain. Included inthe invention are pharmaceutical compositions containing the inventivecompounds and methods of using the compounds as bradykinin receptorantagonists.

More particularly, the invention relates to the modification of thesequence of the mammalian peptide hormone bradykinin(Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) and pharmaceutically acceptablesalts thereof, at the L-Pro residue at position 7 and the L-Phe residueat position 8 in a unique manner which produces sequence-relatedanalogues that act as specific and competitive inhibitors of thebiological activities of bradykinin. The invention specifically relatesto the substitution of the L-Pro at position 7 with D-Phe or D-Tic andat position 8 with a material having the formula:

wherein R is selected from the group consisting of C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkyl substituted C₁-C₆ alkyl, an aryl group, a substituted arylgroup, an arylalkyl group, and a group of the formula R¹NHC(o)— where R¹is C₁-C₆ alkyl or aryl, and where X is sulfur or oxygen;

and pharmaceutically acceptable salts thereof. The * can be either “D”or “L” at the carbon indicated, with the “L” being preferred.

More specifically, the invention relates to the formation of peptideshaving the formula:

N-A-B-C-D-E-F-G-H-I-J-Cn

wherein N is hydrogen;

A and B are independently selected from the group consisting of L-Arg,D-Arg, D-Gln, L-Gln, D-Asn, L-Asn, N-ε-acetyl-D-lysine,ε-acetyl-L-lysine, N^(G)-p-tosyl-Arg, N^(G)-nitro-Arg, Lys-Lys,acetyl-D-Arg, L-Citrulline, L-Lys, D-Lys, and Sar;

C and D are a direct bond or are independently selected from the groupconsisting of Pro, dehydropro, 4Hyp, Tic, Aoc, L-azetidine-2-carboxylicacid, Eac, Gly, Thz, Oic, Aib, and Ala;

E is a direct bond or is selected from the group consisting of Gly, Ala,Thr, and Ser;

F is selected from the group consisting of Phe, Thi, Leu, Ile, Tic, Oic,homoPhe, phenylGly, β-cyclohexylalanine, Nal, and Val;

G is a direct bond or is selected from the group consisting of Ser, Thr,4Hyp, Gly, Val, and Ala;

H is selected from the group consisting of D-Phe and D-Tic;

I is a compound having the formula:

wherein R is selected from the group consisting of C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkyl substituted C₁-C₆ alkyl, an aryl group, a substituted arylgroup, an arylalkyl group, and a group of the formula R¹NHC(o)— where R¹is C₁-C₆ alkyl or aryl, and wherein X is sulfur or oxygen;

J is selected from the group consisting of Arg, Orn, Asn, Gln,N-ε-acetyl-Lys, N-8-acetyl-Orn, and Lys;

Cn is a hydroxyl group or a C-terminal extension selected from the groupconsisting of an amide, alkoxy group, an acidic, basic or neutralaliphatic, aromatic, a cyclic amino acid residue of the D- orL-configuration and a peptide extension composed of D- or L-amino acids;and pharmaceutically acceptable salts thereof.

A particularly preferred material is a peptide wherein:

N is hydrogen;

A and B are independently selected from the group consisting of L-Arg,D-Arg, Lys-Lys, and Lys;

C and D are independently selected from the group consisting of Pro,dehydroPro, and 4Hyp;

E is Gly;

F is selected from the group consisting of Phe, Thi, Leu, andβ-cyclohexylalanine;

G is a direct bond or is selected from the group consisting of Ser, andThr;

H is selected from the group consisting of D-Phe and D-Tic;

I is a compound having the formula:

wherein R is selected from the group consisting of C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkyl substituted C₁-C₆ alkyl, an aryl group, a substituted arylgroup, an arylalkyl group, and a group of the formula R¹NHC(o)— where R¹is C₁-C₆ alkyl or aryl, and where X is sulfur or oxygen;

J is selected from the group consisting of Arg and Lys;

Cn is a hydroxyl group;

and pharmaceutically acceptable salts thereof.

Another preferred material is a peptide wherein:

N is hydrogen;

A is D-Arg;

B is Arg;

C is Pro;

D is selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is selected from the group consisting of Phe, Leu, and Thi;

G is a direct bond or is Ser;

H is selected from the group consisting of D-Phe and D-Tic;

I is a compound having the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, isobutyl, cyclohexylmethyl, alkyl, prenyl, methallyl, benzyl,phenyl, nitrophenyl, and phenylcarbamoyl, and X is sulfur or oxygen;

J is Arg;

Cn is a hydroxyl group;

and pharmaceutically acceptable salts thereof.

The invention may also include some of the intermediate compounds in theL-stereochemical configuration having the formula:

wherein R is selected from the group consisting of C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkyl substituted C₁-C₆ alkyl, an aryl group, a substituted arylgroup, an arylalkyl group, and a group of the formula R¹NHC(o)— where R¹is C₁-C₆ alkyl or aryl, and wherein X is either sulfur or oxygen;

R² is C₁-C₆ alkyl, C₂-C₈ alkenyl, aryl, or arylalkyl;

R³ is H or a suitable amine protecting group.

Preferred peptides according to the invention include the followingnonlimiting materials:

4Hyp Pro Alkyl Ethers

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans methylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans ethylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans propylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans methylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans ethylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans propylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline cis propylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline cis methylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline cis ethylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis ethylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Phe(Hydroxyproline trans methylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Phe(Hydroxyproline trans propylether)-Arg

Pro-Pro Alkyl Ethers

D-Arg-Arg-Pro-Pro-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans methylether)-Arg

D-Arg-Arg-Pro-Pro-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans ethylether)-Arg

D-Arg-Arg-Pro-Pro-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans propylether)-Arg

D-Arg-Arg-Pro-Pro-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans methylether)-Arg

D-Arg-Arg-Pro-Pro-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans ethylether)-Arg

D-Arg-Arg-Pro-Pro-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans propylether)-Arg

Thioalkyl Ethers

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(4-trans thiomethylproline)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(4-trans thioethylproline)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(4-trans thiopropylproline)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(4-trans thiopropylproline)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(4-trans thiomethylproline)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(4-trans thioethylproline)-Arg

Pro 4Hyp Carbamoyl Ethers

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans phenylcarbamoyl)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans phenylcarbamoyl)-Arg

Aryl Ethers and Substituted Aryl Ethers

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis 2-nitrophenylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis 4-nitrophenylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis phenylthioether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis phenylether)-Arg

Thiophenyl Ethers

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(4-trans thiophenylproline)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(4-trans thiophenylproline)-Arg

Allyl Ethers

D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans allylether)-Arg

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans allylether)-Arg

Cycloalkyl Substituted Alkyl Ethers

D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline ciscyclohexylmethyl ether)-Arg

Another embodiment of the invention involves a pharmaceuticalcomposition useful as a bradykinin receptor antagonist comprising apharmaceutical carrier and an effective amount of the novelbradykinin-type peptide. The invention also involves a process forantagonizing bradykinin receptor activity in mammals which comprises:administering to a subject an effective amount of the novel compound toantagonize bradykinin receptor activity.

A further embodiment involves a pharmaceutical preparation for treatinglocal pain and inflammation from burns, wounds, cuts, rashes and othersuch trauma, and pathological conditions caused by the production ofbradykinin or related kinins by an animal which comprises administeringan effective amount of the novel bradykinin type peptide sufficient toantagonize bradykinin with a suitable pharmaceutical carrier. Anotheraspect of this invention involves a process for treating local pain andinflammation which comprises administering an effective amount of thepharmaceutical preparation to an animal in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present compounds which are bradykinin receptor antagonists have thefollowing formula:

N-A-B-C-D-E-F-G-H-I-J-Cn

wherein N is hydrogen;

A and B are independently selected from the group consisting of L-Arg,D-Arg, D-Gln, L-Gln, D-Asn, L-Asn, N-ε-acetyl-D-lysine,ε-acetyl-L-lysine, N^(G)-p-tosyl-Arg, N^(G)-nitro-Arg, Lys-Lys,acetyl-D-Arg, L-citrulline, L-Lys, D-Lys, and Sar;

C and D are a direct bond or are independently selected from the groupconsisting of Pro, dehydropro, 4Hyp, Tic, Aoc, L-azetidine-2-carboxylicacid, Eac, Gly, Thz, Oic, Aib, and Ala;

E is a direct bond or is selected from the group consisting of Gly, Ala,Thr, and Ser;

F is selected from the group consisting of Phe, Thi, Leu, Ile, Tic, Oic,homophe, phenylGly, β-cyclohexylalahine, Nal, and Val;

G is a direct bond or is selected from the group consisting of Ser, Thr,4Hyp, Gly, Val, and Ala;

H is selected from the group consisting of D-Phe and D-Tic;

I is a compound having the formula:

wherein R is selected from the group consisting of C₁-C₆ lower alkyl,substituted C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl,C₃-C₈cycloalkyl substituted C₁-C₆ alkyl, an aryl group, a substitutedaryl group, an arylalkyl group, and a group of the formula R′NHC(o)—where R′ is C₁-C₆ alkyl or aryl and wherein X is sulfur or oxygen;

J is selected from the group consisting of Arg, Orn, Asn, Gin,N-ε-acetyl-Lys, N-δ-acetyl-Orn, and Lys;

Cn is a hydroxyl group a C-terminal extension selected from the groupconsisting of an amide, alkoxy group, an acidic, basic or neutralaliphatic aromatic, a cyclic amino acid residue of the D- orL-configuration and a peptide extension composed of D-or L-amino acids;and pharmaceutically acceptable salts thereof.

Formula 2

Preferred compounds are those in which:

N is hydrogen;

A and B are independently selected from the group consisting of L-Arg,D-Arg, Lys-Lys, and Lys;

C and D are independently selected from the group consisting of Pro,dehydropro, and 4Hyp;

E is Gly;

F is selected from the group consisting of Phe, Thi, Leu, andβ-cyclohexylalanine;

G is a direct bond or is selected from the group consisting of Ser, andThr;

H is selected from the group consisting of D-Phe and D-Tic;

I is a compound having the formula:

wherein R is selected from the group consisting of C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈substituted C₁-C₆ alkyl, an aryl group, a substituted aryl group, anarylalkyl group and X is either sulfur or oxygen;

J is selected from the group consisting of Arg and Lys;

Cn is hydroxyl;

and pharmaceutically acceptable salts thereof.

Formula 3

Most preferred are compounds wherein:

N is hydrogen;

A is D-Arg;

B is Arg;

C is Pro;

D is selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is selected from the group consisting of Phe, Leu, and Thi;

G is a direct bond or is Ser;

H is selected from the group consisting of D-Tic and D-Phe;

I is a compound having the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, isobutyl, cyclohexylmethyl, allyl, prenyl, methallyl, benzyl,phenyl, nitrophenyl, phenylcarbamoyl, and X is sulfur or oxygen;

J is Arg;

Cn is a hydroxyl group; and pharmaceutically acceptable salts thereof.

Formula 4

The inventive compositions also include the following preferredformulations:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is a D-Tic;

I is a compound of the following formula:

wherein R is selected from the group consisting of C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₈ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈substituted C₁-C₆ alkyl, an aryl group, a substituted aryl group, anarylalkyl group and a group of the formula R¹NHC(0)-where R¹ is C₁-C₆alkyl or aryl, and X is either sulfur or oxygen;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 5

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Phe;

I is a compound having the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, phenyl, and nitrophenyl and X is sulfur or oxygen;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 6

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, phenyl, and nitrophenyl and X is sulfur or oxygen;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 7

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is a direct bond;

H is selected from the group consisting of D-Phe and D-Tic;

I is a compound having the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, phenyl, and nitrophenyl and X is sulfur or oxygen;

J is Arg; and pharmaceutically acceptable salts thereof.

Formula 8

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is methyl and X is oxygen;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 9

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is ethyl and X is oxygen;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 10

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is propyl and X is oxygen;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 11

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is phenyl and X is oxygen;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 12

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is allyl and X is oxygen;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 13

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is methyl and X is sulfur;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 14

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is ethyl and X is sulfur;

J is Arg;

and pharmaceutically acceptable salts thereof.

Formula 15

Another preferred formulation is when:

A is D-Arg;

B is Arg;

C and D are selected from the group consisting of Pro and 4Hyp;

E is Gly;

F is Phe;

G is Ser;

H is D-Tic;

I is a compound having the formula:

wherein R is propyl and X is sulfur;

J is Arg;

and pharmaceutically acceptable salts thereof.

As used in the specification and claims, “alkyl” is a paraffinichydrocarbon group which may be derived from an alkane by dropping onehydrogen from the formula such as methyl, ethyl, propyl, isopropyl,butyl, and so forth; “substituted alkyl” is a branched alkyl such asmethyl butyl; “aryl” is an aromatic ring compound such as benzene,phenyl, naphthyl; “substituted aryl” is a substituted aromatic ring suchas nitro substitution, and halogen substitution; and “aralkyl” is anaryl being attached through an alkyl chain, straight or branched,containing from one through six carbons, such as a phenylpropyl group. A“direct bond” is a bond which replaces a particular amino acid compoundwhich may also be between adjacent amino acid and indicated to be absentby the term “molecule”. The phrase “a suitable amino protecting group isa group, such as BOC (t-butyloxycarbonyl-) protecting group whichprotects the amine moiety from reaction and which can be removed undermild conditions so as not to affect the rest of the molecule.

Exemplary Boc protected amino acids include the following nonlimitingmaterials:

N-Boc-L-cis-4-methoxyproline

N-Boc-L-cis-4-ethoxyproline

N-Boc-L-cis-4-(n-propoxy)proline

N-Boc-L-cis-4-phenylthioproline

N-Boc-L-trans-4-methoxyproline

N-Boc-L-trans-4-ethoxyproline

N-Boc-L-trans-4-(n-propoxy)proline

N-Boc-L-trans-4-cyclohexylmethoxyproline

N-Boc-L-trans-4-phenylthioproline

N-Boc-L-cis-4-(4-nitrophenyloxy)proline

N-Boc-L-cis-4-(2-nitrophenyloxy)proline

Definitions of the amino acid abbreviations used herein are as follows:

Arg is arginine; Ala is alanine; Aib is 2 aminoisobutyric acid, Aoc is(S,S,S)-2-azabicyclo[3.3.0]octane-3-carboxylic acid; Asn is asparagine;Eac is E-aminocarproic acid; Gln is glutamine; Gly is glycine; Ile isiscleucine; Leu is leucine; Lys is lysine; Met is methionine; Nal isbeta-2-naphthylalanine; Orn is ornithine; Pro is proline; dehydropro isdehydroproline; homoPhe is homophenylalanine; 4Hyp is 4-hydroxyproline;hydroxyproline is 4-hydroxyproline; Ser is serine; Thi isbeta-2-thienylalanine; Thr is threonine; Thz isthiazolidine-4-carboxylic acid; Phe is phenylalanine; Sar is sarcosine;Tic is tetrahydroisoquinoline-3-carboxylic acid; Oic is (2S, 3aS,7aS)-octahydro-1H-indole-2-carboxylic acid; Val is valine. Furthermore,prenyl is a 3-methyl-2 butenyl radical.

Aoc can be prepared by the method of V. Teetz, R. Geiger and H. Gaul,Tetrahedron Lett. (1984) 4479. Tic can be prepared by the method of K.Hayashi, Y. Ozaki, K. Nunami and N. Yoneda, Chem. Pharm. Bull (1983)31,312.

All amino acids residues, except Gly and Sar, described in thespecification are of the L-configuration unless otherwise specified. TheH position must always be the D-configuration whereas the I position maybe either in the D- or L-configuration with the L-preferred. The symbolsand abbreviations used for amino acids, their derivatives and protectinggroups, and peptides and their salts are those customarily used inpeptide chemistry. (See Biochem. J., (1972) 126, 773, which Journalreference is hereby incorporated by reference).

Table I shows the general location of the amino acid groups as usedherein.

TABLE I N - A - B - C - D - E - F - G - H - I - J - Cn (formula)       Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Bradykinin        1   2   3   4   5   6   7   8   9 (position number)

The synthesis of the peptides of this invention including derivation,activation, and coupling of protected amino acid residues, and theirpurification, and the analytical methods for determining identity andpurity are included in the general body of knowledge of peptidechemistry, as described in Houben Weyl “Methoden der Organischen Chemie”(1974) Vol. 16, parts I & II for solution-phase synthesis, and in SolidPhase Pentide Synthesis (1984) by Stewart and Young for synthesis by thesolid-phase method of Merrifield.

Any chemist skilled in the art of peptide synthesis can synthesize thepeptides of this invention by standard solution methods or by manual orautomated solid phase methods.

The appropriate hydroxyproline substituents used in the 8-position areprepared by the process described in the Examples and depicted in thesequences shown below. The starting materials are commercially availableand/or can be prepared by known procedures. Both the cis and transstereoisomers can be prepared by these means and are within the scope ofthe present invention.

In Scheme II M represents sodium, potassium and other useable salts suchas alkaline earth metals and alkali metals and X is oxygen or sulfur.

Alternately, they also can be prepared by the method of Scheme II fromcommercially available starting materials.

The preparation of compounds for administration in pharmaceuticalpreparations may be performed in a variety of well known methods knownto those skilled in the art. Appropriate pharmaceutically acceptablesalts within the scope of the invention are those derived from mineralacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,nitric acid and sulfuric acid; and organic acids such as tartaric acid,fumaric acid, lactic acid, oxalic acid, ethylsulfonic acid, citric acid,methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, andthe like, giving the hydrochloride, sulfate, phosphate, nitrate,methanesulfonate, tartrate, benzenesulfonate, p-toluensulfonate, and thelike, salt, respectively.

The compounds of the invention may contain an asymmetric carbon atom.Thus, the invention includes the individual stereoisomers, and themixtures thereo. The individual isomers may be prepared or isolated bymethods known in the art.

Therapeutic applications of the novel bradykinin antagonists include notonly treatment for the production of bradykinin or related kinins by theanimal but also the injection of bradykinin related peptides into ananimal as a result of bites and stings. Topical application alone or incombination with subcutaneous utilization of the bradykinin antagonistsof the invention can be employed to treat the effects ofbradykinin-related peptides causing pain, inflammation, and swelling.

The therapeutic use of bradykinin antagonists of this invention forother traumatic inflammatory or pathological conditions which are knownto be mediated by bradykinin or exacerbated by an overproduction ofbradykinin can also be achieved. These conditions include local traumasuch as wounds, burns and rashes, angina, arthritis, asthma, allergies,rhinitis, shock, inflammatory bowel disease, low blood pressure, andsystemic treatment of pain and inflammation.

In parenteral administration of the novel compounds and compositions ofthe invention the compounds may be formulated in aqueous injectionsolutions which may contain antioxidants, buffers, bacteriostats, etc.Extemporaneous injection solutions may be prepared from sterile pills,granules or tablets which may contain diluents, dispersing and surfaceactive agents, binders and lubricants which materials are all well knownto the ordinary skilled artisan.

In the case of oral administration, fine powders or granules of thecompound may be formulated with diluents and dispersing and surfaceactive agents, and may be prepared in water or in a syrup, in capsulesor cachets in the dry state or in a non-aqueous suspension, where asuspending agent may be included. The compounds may also be administeredin tablet form along with optional binders and lubricants, or in asuspension in water or syrup or an oil or in a water/oil emulsion andmay include flavoring, preserving, suspending, thickening andemulsifying agents. The granules or tablets for oral administration maybe coated and other pharmaceutically acceptable agents and formulationsmay be utilized which are all known to those skilled in thepharmaceutical art.

Solid or liquid carriers can also be used. Solid carriers includestarch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc,gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.Liquid carriers include syrup, peanut oil, olive oil, saline, and water.Ointments and creams are prepared using various well known hydrophilicand hydrophobic bases. Topical reservoirs suitably are prepared usingknown polymeric materials such as various acrylic-based polymersselected to provide desired release characteristics. Suppositories areprepared from standard bases such as polyethylene glycol and cocoabutter.

The method of treatment according to this invention comprisesadministering internally or topically to a subject an effective amountof the active compound. Doses of active compounds in the inventivemethod and pharmaceutical compositions containing same are anefficacious, nontoxic quantity selected from the range of 0.01 to 100mg/kg of active compound, preferably 0.1 to 50 mg/kg. Persons skilled inthe art using routine clinical testing are able to determine optimumdoses for the particular ailment being treated. The desired dose isadministered to a subject from 1 to 6 or more times daily, orally,rectally, parenterally, topically, or by inhalation.

The efficacy of the inventive compounds of this invention as bradykininreceptor antagonists can be determined using the bradykinin binding andtissue assays described herein. The results of these assays demonstratethat the novel compounds are potent, selective bradykinin receptorantagonists.

The following examples are illustrative of preferred embodiments ofmethods of preparation and compounds of the invention and are not to beconstrued as limiting the invention thereto.

EXAMPLE 1

This Example demonstrates the preparation ofN-Boc-L-cis-4-methoxyproline by Scheme I.

To a stirred suspension of sodium hydride (3.38 g, 5 80%, 112 mmole)[washed with hexanes, 2×20 mL] in anhydrous dimethylformamide (60 mL)was added dropwise a solution of N-Boc-L-cis-4-hydroxyproline (10.0 g,43.0 mmole) in anhydrous dimethylformamide (60 mL) at room temperature(22° C.) under argon. After 30 min, the suspension was treated withiodomethane (20.0 g, 146 mmole) and the resultant mixture was stirred atroom temperature for 24 hours. Water (100 mL) and aqueous hydrochloricacid (1N) were added until the solution was acidic to the Congo redindicator. The aqueous solution was extracted with diethyl ether (3×250mL), the combined extracts dried over sodium sulfate, and concentratedto an oil. The crude product was used directly in the next step withoutpurification.

To a stirred solution of the crude product in methanol (30 mL) was addedaqueous sodium hydroxide (25 mL, 3 N, 75 mmol) at room temperature (22°C.). After 18 hours, the reaction mixture was diluted with water (35 mL)and concentrated hydrochloric acid was added to adjust the mixture to pH10. The mixture was extracted with diethyl ether (3×55 mL) and theorganic layer was discarded. The aqueous layer was further acidified tothe Congo red indicator endpoint and extracted with ethyl acetate (2×250mL, 1×100 mL). Drying with sodium sulfate and concentration gave an oil.Addition of exane caused precipitation of the product. The solids werecollected, washed with 50% ethyl acetate in hexane (20 mL), and dried invacuo at room temperature to afford the desired product (7.75 g, overallyield 73.3%): mp 119.5-121.8° C.

EXAMPLE 2

This Example demonstrates the preparation ofN-Boc-L-cis-4-n-propoxyproline according to Scheme I.

To a stirred suspension of sodium hydride (2.86 g, 80%, 95.5 mmole)[washed with anhydrous hexane (2×20 mL)] in anhydrous dimethylformamide(60 mL) was added dropwise a solution of N-Boc-L-cis-4-hydroxyproline(8.80 g, 37.8 mmole) in anhydrous dimethylformamide (60 mL) at roomtemperature (22° C.) under argon. After 30 min., a solution of allylbromide (11.46 g, 94.7 mmole) in anhydrous dimethylformamide (35 mL) wasadded dropwise at room temperature. After 24 hours, water (100 mL) wasadded followed by aqueous hydrochloric acid (1 N) until the mixture wasacidic (pH 3). The aqueous solution was extracted with diethyl ether(3×160 mL), the combined extracts were dried over sodium sulfate, andconcentrated to an oil. The crude product was used directly in the nextstep without purification.

To a stirred solution of the crude product in methanol (30 mL) was addedaqueous sodium hydroxide (3 N, 25 mL, 75 mmol) at room temperature.After 18 hours, the reaction mixture was diluted with water (35 mL) andconcentrated hydrochloric acid was added to adjust the solution to pH10. The solution was washed with diethyl ether (2×55 mL) and thecombined organics were discarded. The aqueous layer was acidified to theCongo red indicator endpoint and extracted with ethyl acetate (3×180mL). The combined organics were dried over sodium sulfate andconcentrated to an oil (9.60 g).

A suspension of the above product and 5% platinum on activated carbon(0.74 g) in ethyl acetate (100 mL) was shaken under 35 psi of hydrogenat room temperature. After 6.5 hours, the catalyst was removed andwashed with ethyl acetate. Concentration and flash chromatography(silica gel, 20% methanol in methylene chloride) gave the desiredproduct (8.49 g, overall yield 86.2%) as an oil: IR (neat film) cm−13500-2550 (broad), 2972, 2933, 2877, 1748, 1707, 1478, 1400, 1367, 1164,1100, 1007, 900, 856: 1H NMR (300 MHz, CDCl3) ppm 0.89 (t, 3H, J=7.2Hz), 1.45 (2×s, 9H), 1.55 (q, 2H, J=7.2 Hz), 2.21 (m, 2H), 3.40 (m, 2H),4.04 (t, 1H, J=3.3 Hz), 4.43 (m, 1H), 8.80 (s, 1H)

EXAMPLE 3

This Example demonstrates the preparation ofN-Boc-L-trans-4-n-propoxyproline according to Scheme I.

To a stirred suspension of sodium hydride (1.68 g, 80%, 56.0 mmole)[washed with anhydrous hexane, (2×20 mL)] in anhydrous dimethylformamide(30 mL) was added dropwise a solution of N-Boc-L-trans-4-hydroxyproline(5.0 g, 21.5 mmole) in anhydrous dimethylformamide (35 mL) at roomtemperature (22° C.) under argon. After 30 min, a solution of allylbromide (5.73 g, 47.4 mmole) in anhydrous dimethylformamide (35 mL) wasadded dropwise at room temperature. After 24 hours, the mixture wasdiluted with water (10 mL) and acidified with aqueous hydrochloric acid(5 N) to pH 3. The aqueous solution was extracted with diethyl ether(2×75 mL), and with ethyl acetate (2×75 mL). The combined extracts werewashed with water (2×100 mL), with brine (70 mL), and dried over sodiumsulfate. Concentration gave an oil.

The crude product was used directly in the next step withoutpurification.

A suspension of the crude product (6.0 g) and 5% platinum on activatedcarbon (0.97 g) in ethyl acetate (65 mL) was shaken under 35 psi ofhydrogen at room temperature (22° C.). After 6.5 hours, the catalyst wasremoved and washed with ethyl acetate. Concentration and flashchromatography (silica gel, gradient elution with ethyl acetate inhexane (1:1) to ethyl acetate) gave N-Boc-L-trans-4-n-propoxyprolinepropyl ester (2.90 g) as an oil.

To a stirred solution of N-Boc-L-trans-4-hydroxyproline propyl etherpropyl ester (2.90 g, 9.16 mmole) in ethanol (10 mL) was added aqueoussodium hydroxide (12 mL, 3 N, 36 mmol) at room temperature. After 4hours, the reaction mixture was acidified to the Congo red indicatorendpoint with aqueous hydrochloric cid (3 N), the reaction mixture wassaturated with sodium chloride, and extracted with diethyl ether (4×45mL). The combined organics were dried over sodium sulfate andconcentrated to an oil. Flash chromatography (silica gel, methylenechloride: methanol: acetic acid 90:8:2) gave the desired product (2.50g, overall. yield 42.5%) as an oil: ¹H NMR (300 MHz, CDCl3) ppm 0.91(t,3H, J=7.5); 1.45 (2×s, 9H), 1.56 (m, 2H), 2, 28 (t, 1H, J=6.6 Hz), 2.37(m, 1H), 3.40 (m, 2H), 3.55 (m, 2H), 4.06 (q, 1H, J=4.5 Hz), 4.38 (m,1H), 10.54 (s, 1H).

EXAMPLE 4

This Example demonstrates the preparation ofN-Boc-L-cis-4-ethoxyproline.

To a stirred suspension of sodium hydride (1.94 g 80% 64.8 mmol) inanhydrous dimethylformamide (100 mL) was added in small portionsN-Boc-L-cis-4-hydroxyproline (6.0 g 26 mmol) at room temperature underargon. After 30 min the suspension was treated with iodoethane (5.20 mL54.5 mmol) at room temperature. After 27 hours, the reaction mixture wasacidified with aqueous hydrochloric acid solution at the Congo redindicator endpoint and saturated brine, dried over sodium sulfate, andconcentrated to an oil which was used directly in the next step withoutpurification.

To a stirred solution of the product in methanol (25 mL) was addedaqueous sodium hydroxide solution (20 mL, 3N, 60 mmol) at roomtemperature. After stirring 6 hours, water was added and the mixtureextracted with diethyl ether (2×20 mL). The organic extracts werediscarded. The aqueous layer was acidified to the Congo red indicatorendpoint and extracted with ethyl acetate (3×100 mL). The combinedextracts were dried over sodium sulfate. Flash chromatography (silicagel, 15% methanol in dichloromethane) gave the desired product (5.50 g,68.4%) as a solid: mp 53-56.2° C.; IR (KBr) cm⁻¹ 3500-2500, 1723, 1622,1434, 1250, 1095, 897, 848, 769; ¹H NMR (300 MHz, CDCl₃)ppm 1.18 (t, 3H, J=6.6Hz), 1.46 (s, 9 H), 2.32 (m, 2H), 3.51 (m, 4H), 4.06 (m, 1H),4.37 (m, 1H), 8.20 (s, 1H).

Dicyclohexylamine salt (recrystallized from heptane): mp 161-162.4° C.;[α]^(21.5) _(D)=−33.5 (c=0.98, methanol). Anal. Calcd for C₂₄H₄₄N₂O₅(440.62 g/mol): C, 65.42;; H, 10.07; N, 6.36. Found: C, 65.32; H, 10.05;N, 20 6.37.

EXAMPLE 5

This example demonstrates the preparation ofN-Boc-L-trans-4-methoxyproline.

A stirred suspension of sodium hydride (1.43 g, 80%, 47.7 mmol) [washedtwice with hexanes] in a mixture of anhydrous N,N-dimethylformamide (15mL) and anhydrous tetrahydrofuran (40 mL) at 5° C. under argon was addedN-Boc-L-trans-4-hydroxyproline (5.00 g, 21.6 mmol). When the gasevolution had subsided (ca. 10 min), iodomethane (3.40 mL, 54.1 mmol)was added at 5° C. After 24 hours at room temperature, the suspensionwas diluted with water (30 mL) and acidified to the Congo red indicatorendpoint with aqueous hydrochloric acid (1N) and extracted with ethylacetate (4×100 mL). The combined organics were washed with aqueoussodium thiosulfate, with water, with brine, and dried (magnesiumsulfate) Concentration gave a yellow oil which was used in the next stepwithout purification.

To a stirred solution of the oil in water (25 mL) and 2-propanol (8 mL)was added aqueous potassium hydroxide (16.5 mL, 2.0 N, 33 mmol). After 5days at room temperature, the mixture was diluted with water (10 mL) andextracted with diethyl ether (2×50 mL). The combined organics wereback-extracted with half-saturated aqueous potassium bicarbonate (20 mL)and discarded. The combined aqueous layers were cooled to 5° C.,acidified to pH 4 with citric acid, saturated with sodium chloride, andextracted with ethyl acetate (4×50 mL). The combined ethyl acetateextracts were dried (sodium sulfate) and concentrated to an oil. Flashchromatography (silica gel, 91:8:1 chloroform: methanol: acetic acid)followed by extensive drying in vacuo gave the desired product as aslightly yellow syrup (4.90 g, 92% overall)): IR (KBr) cm⁻¹ 2977, 2933,1746 (sh), 1697, 1417, 1368, 1254, 1162, 1098: ¹H NMR (300 MHz, CDCl₃)ppm 1.42 & 1.47 (2×s, 9H total), 2.10 (m, 1H), 2.24 (m, 1H), 3.33 (s,3H), 3.60 (m, 2H), 4.00 (m, 1H), 4.30 & 4.42 (2×m, 1H total), 10.27 (brs, 1H).

Dicyclohexylammonium salt (recrystallized from n-heptane): mp 126-128°C.; [α]^(22.5) _(D)=−30.5 (c=1.02, methanol). Anal. for C₂₃H₄₂N₂O₅(426.60 g/mol): C, 64.76; H, 9.92; N, 6.57. Found: C, 64.68; H, 9.96; N,6.53.

Cyclohexylammonium salt (recrystallized from ethyl acetate): mp 155-158°C.; {α]^(22.5) _(D)=−38.7 (c=1.01, methanol). Anal, Calcd for C₁₇H₃₂N₂O₅(344.45 g/mol): C, 59.28; H, 9.36; N, 8.13. Found C, 59.02; H, 9.38; N,8.09.

EXAMPLE 6

This Example demonstrates the preparation ofN-Boc-D-trans-4-phenylthioproline according to Scheme II.

To a stirred suspension of hexane washed sodium hydride (3.06 g, 80%,38.1 mmol) in anhydrous tetrahydrofuran (95 mL) was added dropwisethiophenol (4.50 mL, 43.7 mmol) at room temperature (22° C.) underargon. After 1 hour, the mixture was treated withN-Boc-D-cis-4-(p-toluenesulfonyloxy)proline (5.00 g, 12.5 mmol) at roomtemperature. The resultant mixture was heated to reflux for 8 hours.After cooling to room temperature, the mixture was acidified to theCongo red indicator endpoint with aqueous hydrochloric acid. Thesolution was extracted with ethyl acetate (4×80 mL) and the combinedextracts were dried over sodium sulfate. Concentration gave an oil whichwas used directly in the next step without purification.

To a stirred solution of the crude N-Boc-D-trans-4-phenylthioprolinemethyl ester in methanol (20 mL) at room temperature was added asolution of sodium hydroxide (3N, 18 mL). After two days at roomtemperature, water (30 mL) was added and the mixture was extracted withdiethyl ether (3×45 mL). The combined organics were discarded and theaqueous layer was acidified with aqueous hydrochloric acid (5 N) to theCongo red indicator endpoint. The aqueous layer was extracted with ethylacetate (3×110 mL) and the combined extracts were dried over sodiumsulfate. Concentration followed by flash chromatography (silica gel,methylene chloride/methanol/acetic acid 90:8:2) gaveN-Doc-D-trans-4-phenylthio-proline (3.64 g, 79.3%) as an oil: IR (neatfilm) cm⁻¹ 3300-2500, 1749, 1702, 1583 (w), 1415, 1398, 1368, 1164, 743;¹H NMR (300 MHz, CDCl₃) ppm 1.45 & 1.48 (2×s, 9H), 2.31 (m, 1H), 3.44(m, 1H), 3.76 (m, 2H), 4.43 (m, 1H), 7.37 (m, 3H), 7.42 (m, 2H), 9.77(s, 1H),

EXAMPLE 7

This Example demonstrates the preparation ofN-Boc-L-trans-4-ethoxyproline.

To a stirred suspension of hexane washed scdium hydride (1.56 g, 80%,51.9 mmol) in anhydrous tetrahydrofuran (100 mL) was added in smallportions N-Boc-L-trans-4-hydroxyproline (6.00 g, 25.9 mmol) at roomtemperature and under argon. After 1 hour, the suspension was treatedwith iodoethane (4.15 mL, 51.9 mmol) at room temperature. The reactionmixture was heated to reflux for 3 hours then cooled to room temperatureand stirred overnight. The reaction mixture was diluted with water andextracted with hexane (30 mL). The hexane extract was discarded. Theaqueous layer was acidified with concentrated hydrochloric acid to theCongo red indicator endpoint and extracted with ethyl acetate (3×120mL). The combined extracts were dried over sodium sulfate. Flashchromatography (silica gel, methanol: dichloromethane: acetic acid10:90:1) gave the desired product (4.98 g, 74.2%) as a solid: mp48.5-51.2° C.; IR (KBr) cm⁻¹ 3500-2600, 1738, 1640, 1434, 1367, 1244,1172, 1100, 771; ¹H NMR (300 MHz, CDCl₃) ppm 1.20 (t, 3 H, J=6.9 Hz),1.42 & 1.48 (2×s, 9 H), 2.25 (m, 2H), 3.51 (m, 4H), 4.08 (m, 1H), 4.35(t, 1/2H, J=7.8 Hz), 4.44 (m, 1/2H), 9.06 (s, 1H).

Dicyclohexylamine salt (recrystallized from heptane): mp 128.5-130.5°C.; [α]^(21.5) _(D)=−30.2 (c=1.02, methanol). Anal. Calcd for C₂₄H₄₄N₂O₄(440.62 g/mol): C, 65.42; H, 10.07; N, 6.36. Found C, 65.31; H, 10.02;N, 6.38.

EXAMPLE 8

This Example demonstrates the preparation ofN-Boc-L-cis-4-phenylthioproline.

To a stirred suspension of hexane washed sodium hydride (1.61 g, 80%,53.6 mmol) in anhydrous tetrahydrofuran (120 mL) was added thiophenol(6.30 mL, 61.3 mmol) dropwise at room temperature under argon. After 1hour, the mixture was treated withN-Boc-L-trans-4-(p-toluenesolfonyloxy)proline methyl ester (5.00 g, 12.5mmol) at room temperature. The resultant mixture was heated to refluxfor 7.5 hours, then cooled to room temperature and stirred overnight.The mixture was acidified to the Congo red indicator endpoint withaqueous hydrochloric acid and the layers were separated. The aqueouslayer was extracted with ethyl acetate (3×100 mL) and the combinedorganics were dried over sodium sulfate. Concentration gave an oil whichwas used directly in the next step without purification.

To a stirred solution of the crude N-Boc-L-cis-4-phenylthioprolinemethyl ester in methanol (25 mL) at 5° C. was added aqueous sodiumhydroxide (15 mL, 3 N, 45 mmol). The mixture was allowed gradually towarm to room temperature. After 18 hours at room temperature, water wasadded and the mixture was extracted with hexane (2×25 mL). The combinedorganics were discarded and the aqueous layer was acidified with aqueoushydrochloric acid (5 N) to the Congo red indicator endpoint. The aqueouslayer was extracted with ethyl acetate (3×120 mL) and the combinedextracts dried over sodium sulfate. Concentration followed by flashchromatography (silica gel, dichloromethane/methanol/acetic acid90:10:1) gave the desired product (5.60 g, 98.8%) as a white ,hygroscopic solid: mp 76-79.5° C.; IR (neat film) cm⁻¹3500-2500, 1699(br), 1584 (w), 1478, 1416, 1159, 743, 691; H NMR (300 MHz, CDCl₃)ppm1.42 & 1.47 (2×s, 9H), 2.22 (m, 1H), 2.65 (m, 1H), 3.38 (m, 1H), 3.66(m, H), 3.92 (m, 1H), 4.34 (m, 1H), 7.29 (m, 3H), 7.42 (d, 2H, J=6.6Hz), 9.45 (s, 1H).

Dicyclohexylamine salt (recrystallized from acetonitrile): mp 168-169.2°C.; [α]²³ _(D)=−44.2 (c=1.03, methanol). Anal. Calcd for C₂₈H₄₄N2O₄S(504.73 g/mol): C, 66.63; H, 8.79; N, 5.55. Found C, 66.56; H, 8.82; H,15 5.53.

EXAMPLE 9

This Example demonstrates the preparation ofN-Boc-L-trans-4-phenylthioproline.

To a stirred suspension of hexane washed sodium hydride (1.35 g, 80%,45.1 mmol) in anhydrous tetrahydrofuran (90 mL) was added thiophenol(3.90 mL, 38.0 mmol) Boc-L-cis-4-(p-toluenesulfonyloxy)proline methylester (10.0 g, 25.0 mmol) at room temperature. The resultant mixture washeated to reflux for 5 hours, then stirred at room temperatureovernight. After 16 hours, the mixture was diluted with water andacidified to the Congo red indicator endpoint with aqueous hydrochloricacid (SN). The solution was extracted with thyl acetate (3×100 mL) andthe combined extracts ried over sodium sulfate. Concentration gave anoil hich was used directly in the next step without purification.

To a stirred solution of the crude N-Boc-L-trans-4-phenylthioprolinemethyl ester in methanol (30 mL) at room temperature was added aqueoussodium hydroxide (18.0 mL, 3N, 54 mmol). After 18 hours, water was addedand the mixture was extracted with diethyl ether (3×20 mL). The combinedorganics were discarded and the aqueous layer was acidified with aqueoushydrochloric acid (5 N) to the Congo red indicator endpoint. The aqueouslayer was extracted with ethyl acetate (4×120 mL) and the combinedextracts dried over sodium sulfate. Concentration gave a yellow oilwhich was used in the next step without purification.

The oil was dissolved in acetonitrile at room temperature and treatedwith cyclohexylamine (3.30 mL, 28.8 mmol). The precipitated solid wasrecrystallized in the same solvent. The crystalline product wasdissolved in water and acidified with aqueous hydrochloric acid (5 N) tothe Congo red indicator endpoint. The aqueous layer was extracted withethyl acetate (3×120 mL) and the combined extracts dried over sodiumsulfate.

Concentration gave N-Boc-L-trans-4-phenylthioproline (6.85 g, 85%) as afoam: IR (film) cm⁻¹ 3300-2500, 1749, 1702, 1583, 1415, 1398, 1368,1164, 743; ¹H NMR (300 MHz, CDCl₃) ppm 1.45 & 1.48 (2×s, 9H), 2.31 (m,2H), 3.44 (m, 1H), 3.76 (m, 2H), 4.43 (m, 1 H), 7.36 (m, 3H), 7.42 (m,2H), 9.77 (s, 1H); [α]^(23.5) _(D)=−26.9 (c 2.04, methanol).

Dicyclohexylamine salt (recrystallized from acetonitrile): mp164.5-165.8° C.; [α]^(23.5) _(D)=−16.0 (c 1.00, methanol). Anal. Calcdfor C₂₈H₄₄N₂O₄S (504.73 g/mol): C, 66.64; H, 8.79; N, 5.55. Found: C,66.65; H, 8.81; H, 5.57.

Cyclohexylamine salt (recrystallized from acetonitrile): mp 170-172.5°C.; [α]^(23.5) _(D)=−18.5 (c 1.02, methanol). Anal. Calcd forC₂₂H₃₄N₂O₄S (422.58 g/mol): C, 62.53; H, 8.11; N, 6.63. Found: C, 62.52;H, 8.13; N, 6.62.

EXAMPLE 10

This Example demonstrates the preparation of (2S,4R)-N-(tert-Butoxycarbonyl)-4-O-(phenylcarbamoyl)proline.

To a stirred solution of N-Boc-L-trans-hydroxyproline methyl ester (4.05g, 16.5 mmol) and 4-dimethylaminopyridine (0.11 g, 0.89 mmol) in CHCl₃(30 mL) was added phenyl isocyanate (1.82 mL, 16.7 mnol) at roomtemperature. After 21 hours, the mixture was washed with aqueous HCl (10mL, 0.5 N) and dried (MgSO₄). Concentration and drying in vacuo gave(2S, 4R)-N-Boc-4-O-(phenylcarbamoyl)proline methyl ester (6.00 g, 100%)as white solids: mp 129-131° C.

The a stirred suspension of this ester (5.00 g, 14.4 mmol) in MeOH (20mL) and water (5 mL) was added aqueous NaOH (5.0 mL, 3 N, 15 mmol).After 20 hours at room temperature, additional aqueous NaOH (1.0 mL, 3N,3.0 mmol) was added. After an additional 4 hours, the mixture wasextracted with EtOAc (3×30 mL). The organics were discarded and theaqueous layer was acidified to the Congo red indicator endpoint withconcentrated HCl at 5° C. The mixture was saturated with NaCl andextracted with EtOAc (4×50 mL). The combined organics were dried(Na₂SO₄) and concentrated to give white solids which were purified byflash chromatography (silica gel, 90:10:1 CH₂Cl₂:MeOH:HOAc) to affordthe desired product (3.66 g, 76%) as white solids: mp 161-163° C.; IR(KBr) cm⁻¹ 3430, 3247, 1730, 1686, 1607, 1550, 1445, 1419, 1226, 1159,1069, 753; ¹H NMR (300 MHz, CDCl₃) δ 1.44 & 1.47 (2×s, 9H), 2.38 (m,1H), 2.49 (m, 1H), 3.70 (m, 2H), 4.41 (m, 1H), 5.33 (m, 1H), 7.07 (m,2H), 7.30 (m, 3H), 7.38 (br s, 1H), 9.67 (br s, 1H); [α]^(22.5)_(D)=−38.9 (c=1.05, MeOH). Anal. Calcd for C₁₇H₂₂N₂O₆. 0.75 H₂O (363.88g/mol): C, 56.11; H, 6.51; N, 7.70. Found: C, 56.20 & 56.13; H, 6.49 &6.51; N, 7.75.

General Procedure for Synthesis of Nitrophenyl Ethers of Hydroxyproline

To a warm (40°-60° C.) solution of powdered KOH (8.69 g, 158 mmol) inabsolute ethanol (240 mL) was added a solution of 2- or 4-nitrophenol in120 mL of acetone.

The resulting suspension was stirred vigorously and heated to reflux. Atreflux a solution of Boc-4Hyp(Tosyl)-OMe (30.0 g, 63.2 mmol) in 120 mLof acetone was added dropwise during one hour. The mixture was allowedto stir at reflux for the number of days given below for each specificcompound. At the end of this time, the precipitate which had formed wasfiltered off and washed with acetone. The filtrate and washing wereevaporated at reduced pressure and the residue from evaporation wasdiluted with 750 mL of water. The aqueous suspension was extracted threetimes with 200 mL portions of CH₂Cl₂. The extracts were combined andsplashed three times with 200 mL portions of 5% aqueous NaOH. The CH₂Cl₂solution was washed once with 200 mL of saturated aqueous NaHCO₃, washedwith 200 mL of brine, dried over MgSO₄, filtered, evaporated underreduced pressure, and stored in vacuo.

The compounds below were synthesized using the general procedure butwith appropriately scaled amounts of reagents.

EXAMPLE 11

This Example demonstrates the preparation ofN-Boc-L-cHyp(2-nitrophenyloxy)-OMe

From 2.0 g (3.0 mmol) of N-Boc-L-cHyp(Tosyloxy)-OMe and 2.5 equivalentseach of KOH (0.42 g, 7.5 mmol) and 2-nitrophenol (1.04 g, 7.5 mmol) atreflux for 6 days was obtained 1.16 g (82%) ofN-Boc-L-cHyp(2-nitrophenyloxy)-OMe as an oil after purification bycolumn chromatography on silica gel with ethyl acetate. The purifiednitrophenyl hydroxyproline ether was about 60% ethyl and 40% methylester, due to the long reaction time. The two esters were not separable.

IR(neat): v 3583, 3499, 3978, 1751, 1700, 16.07, 5.27, 1484, 1401, 1365,1281, 1255, 1201, 1170, 1071, 1067, 905, 851, 771, 746, 661 Cm⁻¹.

NMR(CDCl₃): δ 1.214-1.281 (m, 2.5H), 1.415-1.455 (2s, 9H), 2.551-2.590(m, 2H), 3.701-3.852 (m, 3.2H), 4.086-4.449 (m, 4H), 6.928-6.956 (d, 1H,J=8.30 Hz), 7.045 (m, 1H), 7.486-7.510 (m, 1H), 7.782-7.809 (d, 1H,J=8.06).

EXAMPLE 12

This Example demonstrates the preparation ofN-Boc-L-cHyp(4-nitrophenyloxy)-OMe

Using N-Boc-L-tHyp(Tosyloxy)-OMe (5g, 10.5 mmol), powdered KOH (1.75 g,26.5 mmol) and 4-nitrophenol (3.66 g, 26.3 mmol) after 4 days at reflux,2.52 g (65%) of the corresponding 4-nitrophenyl ether was obtained bycolumn chromatography (silica ge, ethyl acetate/hexane mixtures) andcrystallization of the appropriate fractions from ethyl acetate/hexane(1:1). An alternate method is to triturate the oily crude and pentaneseveral times [hexane doesn't work] and then add a very, small amount ofethyl acetate to solidify the product. The filtered, solid product maythan be crystallized as above. Elemental analysis for C₁₇H₂₂N₂O₇(366.324 g/mmol): Calculated C, 55.74; H, 6.05; N, 7.65. Found C, 55.71;H, 6.05; N, 7.65.

IR(neat): V 3111, 3080, 2978, 1756, 1733, 1694, 1607, 1594, 1512, 1494,1399, 1363, 1337, 1260, 1198, 1173, 1160, 1121, 1108, 1062, 962, 900,890, 846, 751, 689, 648 cm⁻¹.

EXAMPLE 13

This Example demonstrates the preparation ofN-Boc-D-tHyp(2-nitrophenyloxy)-OMe.

From N-Boc-D-cHyp(Tosyloxy)-OMe (5.27 mmol, 2.50 g), powdered KOH (13.2mmol, 0.74 g), and 4-nitrophenol (13.2 mmol, 1.83 g), held at reflux for6 days, the 4-nitrophenyl ether of D-trans-hydroxyproline methyl ester(0.39 g, 20%, m.p. 154-155° C.) was obtained after purification bycolumn chromatography (silica gel, ethyl acetate/hexane mixtures).Impure fractions (1.14 g) accounted for the low yield relative toL-hydroxyproline derivatives above. Elemental analysis for C₁₇H₂₂N₂O₇:Calculated C, 55.74; H, 6.05; N, 7.65. Found: Calculated C, 55.71; H,6.08; N, 7.69.

EXAMPLE 14

This Example demonstrates the preparation ofN-Boc-D-tHyp(4-nitrophenyloxy)-OMe.

From N-Boc-D-cHyp(Tosyloxy)-OMe (5.27 mmol, 2.50 g), powdered KOH (13.2mmol, 0.74 g), and 2-nitrophenol (13.2 mmol, 1.83 g), held at reflux for6 days, the 2-nitrophenyl ether of D-trans-hydroxyproline methyl ester(0.29 g, 14%) was obtained. Purification by column chromatography(silica ge, ethyl acetate/hexane mixtures) yielded no pure fractions.All fractions bearing product were combined and purified in three equalportions using the chromatotron (4 mm silica gel plates) and a mixtureof ethyl acetate/CH₂Cl₂/hexane (2:9:9:) which was found to effectseparation on TLC plates. The mixture of ethyl acetate/CH₂Cl₂ (2:9) willbe referred to as mixture A or simply A.

Chromatotron separation procedure: A sample of the impure product (1.0g) was dissolved in A and applied to the chromatotron plate wetted andequilibrated with A/hexane (1:30) followed by a hexane wash. Whilemonitoring the migration of the impurities (fast bands) vs. product(slowest band) by UV light, the amount of A was gradually increased inthe mixtures by reducing the amount of hexane in increments of 5 parts.The volume of each mixture was 200 mL. Fractions of 5-10 mL werecollected when the product band seemed to be eluting. When the ratio ofA to hexane reached 1:10, 200 mL of 1:9 was applied and the plate waspolarized with ethyl acetate/CH₂Cl₂/hexane (1:4:4). The fractionsbearing pure product were concentrated to a yellow oil which solidifiedseveral days later after the side of the glass container was scratched.

Elemental analysis for C₁₇H₂₂N₂O₇: Calculated C, 55.74; H, 6.05; N,7.65. Found: Calculated C, 55.67; H, 6.12; H, 7.50.

IR(neat): V 3114, 3083, 2978, 2933, 1749, 1700, 1609, 1594, 1515, 1497,1401, 1368, 1342, 1257, 1209, 1162, 1129, 1113, 1072, 905, 849, 753,692, 645, 609 cm⁻¹.

NMR(CDCl₃: δ1.484-1.479 (2s, 9H), 2.518 (br s and m, 2H total),3.672-3.886 (m, 5H), 4.451-4.488 (dd, 0.6H, J_(A)=2.51 Hz, J_(B)=8.48Hz), 4.588 (t, 0.4H, J=8.56 Hz), 5.005-5.018 (m, 2H), 6.854-6.881 (d,1H, J=8.31 Hz), 8.180-8.207 (d, 2H, J=8.08 Hz).

EXAMPLE 15

This Example demonstrates the preparation of N-Boc-(2S, 3aS,7aS)-Octahydro-1H-indole-2-carboxylic Acid (Oic).

A mixture of (S)-indoline-2-carboxylic acid (10.01 g, 60.73 mmol) and10% platinum on activated carbon (0.57 g) in aqueous hydrochloric acid(150 Ml, 1 N, 150 mmol) and ethanol (20 Ml) was shaken in a Parr bottleunder 45 psi of hydrogen at room temperature (22° C.). After 20 hours,the mixture was filtered and the solids washed with methanol. Thecombined filtrates were concentrated to 50 Ml and the solution treatedwith sodium carbonate (9.65 g, 91 mmol) di-tert-butyl dicarbonate (17.5Ml, 77.4 mmol) and 20 Ml of dioxane. The mixture was stirred for 18hours, diluted with water (50 Ml), and the mixture extracted with ethylether (3×30 Ml). The aqueous layer was decolorized with charcoal,acidified to the Congo red indicator endpoint with concentratedhydrochloric acid, saturated with sodium chloride and extracted withethyl acetate (5×50 Ml). The combined extracts were dried (sodiumsulfate) and the solvent removed to give a white foam. Recrystallizationfrom heptane gave the carboxylic acid (11.51 g, 70%) as white crystals:mp 129-131° C.

General Procedure for Automated Peptide Synthesis:

Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Thi-D-Tic-(hydroxyproline cispropyl ether)-Arg

The peptide was synthesized employing t-Boc chemistry on a solid phasesynthesizer (Milligen Biosearch 9600 Peptide Synthesizer).Boc-Arg(Tos)-PAM resin (Applied Biosystems) (PAM=phenylacetamidomethyl),0.25 g, with a resin substitution of 0.62 mmol Arg/gram of resin, wasplaced in the reaction vessel and subjected to Procedure A for thecoupling of Boc-Oic. Commercially available amino acids were purchasedfrom Bachem Bioscience. Volumes of reagents and solvents wereapproximately 20 ml/gram of resin.

Procedure A:

1. Deprotection: Removal of the t-butyloxycarbonyl-protecting group(Boc) was achieved by treatment of the resin with deblocking reagent(trifluoroacetic acid (TFA)/anisole/dichloromethane(DCM) 45:2.5:52.5 v/vcontaining 1 mg/Ml of indole), two times for one minute and once fortwenty minutes. The resin was then washed with DCM several times,followed by neutralization with base [10% diisopropylethylamine (DIEA)in DCM], three times for one minute. The resin was subsequently washedwith DCM and dimethylformamide (DMF).

2. Coupling: All couplings and recouplings were mediated in the samemanner. Boc-Oic (1.47 mmol, 0.4 M in DMF) was mixed with one equivalentof diisopropylcarbodiimide (DIPCDI) (1.47 mmol, 0.4 M in DCM) for a twominute activation period prior to coupling with the resin. The mixturewas added to the reaction vessel containing the resin and mixed for twohours. Coupling efficiency of the amino acid to the growing peptidechain on the resin was checked. Incomplete coupling of an amino acidresulted in a recoupling step. Recoupling involved washing theresin-peptide three times for one minute with base followed by DCM andDMF. Amino acid activation with DIPCDI with addition to thepeptide-resin was repeated and allowed to mix an additional two hours.After a successful coupling the peptide-resin was washed several timeswith DCM.

3. Capping: The growing peptide chain was capped on the α-amino group byacetylation with 1-acetylimidazole (0.3 M in DMF) at the end of eachcoupling or recoupling. The resin was washed three times with basefollowed by DCM and DMF. The resin was treated with capping reagent for30 minutes and then washed with DMF.

Procedure B:

The N-terminal protecting group was removed by the following procedure:

Terminal deprotection: Following the capping of the final amino acid tobe added to the growing peptide chain, the peptide-resin was treatedwith deblocking reagent (TFA/anisole/DCM) twice for one minute and oncefor 20 minutes. The resin was washed with DCM followed by methanol andthen dried by a stream of inert gas.

The following amino acids were added to the growing peptide chainaccording to the listed programs: Boc-D-Phe (A), Boc-Ser(Bzl) (A),Boc-Thi (A), Boc-Gly (A), Boc-Hyp(Bzl) (A), Boc-Pro (A), Boc-Arg(Tos)(A), Boc-D-Arg(Tos) (A),(B). This yielded 0.481 g of protectedpeptide-resin as the TFA salt.

HF Cleavage: The peptide-resin (0.481 g) was suspended in 5 Ml of liquidanhydrous HF (ratio of 10 Ml HF/g resin) containing 0.48 Ml of anisoleat −70° C. and stirred for 60 minutes at 0° C. The HF was removed by astream of nitrogen gas followed by vacuum (water aspirator). The resinwas washed three times with 30 Ml of ethyl ether and dried under highvacuum for 30 minutes. The peptide was extracted with distilleddeionized water (200 Ml) and the solution was lyophilized to give 176 mgof crude deprotected peptide.

Purification: The peptide was purified on a reverse phase C-18 (2×25 cm)Vydac HPLC column using a gradient of 0.1% TFA/H2O and acetonitrile(0.1% TFA) to give 53 mg of purified deprotected peptide.

Analysis: Purified peptide was characterized by amino acid analysis andgave the following results: Arg, 2.9 (3.0); Ser, 0.92 (1.0); Thi, 1.09(1.0); Gly, 1.0 (1.0).

The peptide was also characterized by mass spectrometry (JEOL HX110/110FAB) [M+H] obsd 1308.7, [M+H] calcd 1308.6.

EXAMPLE 16

Using the method of Example 15, the peptideD-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(hydroxyproline cis methylether)-Arg was prepared. Purified peptide was characterized by aminoacid analysis and gave the following results: Arg, 3.38 (3.0); Ser, 0.84(1.0); Thi, 1.14 (1.0); Gly, 1.0 (1.0). The peptide was alsocharacterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd1280.7, [M+H] calcd 1280.6

EXAMPLE 17

Using the method of Example 15, the peptideD-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans propylether)-Arg was prepared from the appropriate amino acids. Purifiedpeptide was characterized by amino acid analysis and by massspectrometry (JEOL HX110/110 FAB).

EXAMPLE 18

Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis2-nitrophenyl ether)-Arg

Using the method of Example 15, the peptideD-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis 2-nitrophenylether)-Arg was prepared. Purified peptide was characterized by aminoacid analysis and gave the following results: Arg, 2.57 (3.0); Ser, 1.01(1.0); Phe, 0.94 (1.0); Gly, 1.0 (1.0). The peptide was alsocharacterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd1381.77, [M+H] calcd 1381.7.

EXAMPLE 19

Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis4-nitrophenyl ether)-Arg.

Using the method of Example 15, the peptideD-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis 4-nitrophenylether)-Arg was prepared. Purified peptide was characterized by aminoacid analysis and gave the following results: Arg, 3.15 (3.0); Ser. 0.94(1.0); Phe, 0.98 (1.0); Gly, 1.0 (1.0). The peptide was alsocharacterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd1381.98, [M+H] calcd 1381.7.

EXAMPLE 20

Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cisethyl ether)-Arg.

Using the method of Example 15, the peptideD-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis ethylether)-Arg was prepared. Purified peptide was characterized by aminoacid analysis and gave the following results: Arg, 3.15 (3.0); Ser, 0.87(1.0); Phe, 1.05 (1.0); Gly, 1.0 (1.0). The peptide was alsocharacterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd1288.17, [M+H] calcd 1288.68.

EXAMPLE 21

Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline ciscyclohexylmethyl ether)-Arg.

Using the method of Example 15, the peptideD-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline ciscyclohexylmethyl ether)-Arg was prepared. Purified peptide wascharacterized by amino acid analysis and gave the following results:Arg, 2.83 (3.0); Ser, 0.98 (1.0); Phe, 1.03 (1.0); Gly, 1.0 (1.0). Thepeptide was also characterized by mass spectrometry (JEOL HX110/110 FAB)[M+H] obsd 1357.08, [M+H] calcd 1356.7.

EXAMPLE 22

Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cisphenyl thioether)-Arg.

Using the method of Example 15, the peptideD-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis phenylthioether)-Arg was prepared. Purified peptide was characterized by aminoacid analysis and gave the following results: Arg, 2.51 (3.0); Ser, 0.78(1.0); Phe, 1.0 (1.0). The peptide was also characterized by massspectrometry (JEOL HX110/10 FAB) [M+H] obsd 1352.73, [M+H] calcd 1352.7.

EXAMPLE 23

Preparation of D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cisphenyl ether)-Arg.

Using the method of Example 15, the peptideD-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(hydroxyproline cis phenylether)-Arg was prepared. Purified peptide was characterized by aminoacid analysis and gave the following results: Arg, 2.69 (3.0); Ser, 0.89(1.0); Phe, 1.05 (1.0); Gly, 1.0 (1.0). The peptide was alsocharacterized by mass spectrometry (JEOL HX110/110 FAB) [M+H] obsd1336.79, [M+H] calcd 1336.7.

Bradykinin Binding Procedure

Binding of ³H-Bradykinin was preformed using the method of D. C.Manning, R. Vavrek, J. M. Stewart, and S. H. Synder, J. Pharmacol. Exp.Ther., (1986),237 504. The tissues used in the binding assay wereterminal ileum from male Hartley guinea pigs (150-350 g). Afterdissection, tissues were placed in 20 vol of ice-cold buffer A (25 MmTES containing 0.2 g/L of 1,10-phenanthroline adjusted to Ph 6.8 withammonium hydroxide) and homogenized using a Ploytron Tissumizer atsetting 6 for 15 sec. The homogenate was centrifuged at 50,000×g for 10min, the supernatant discarded, and the pellet resuspended in ice-coldbuffer A by homogenization with the Polytron. Each tissue washomogenized and centrifuged three times. The final pellet wasresuspended in buffer A containing bovine serum albumin (1 g/L) andBacitracin (0.14 g/L) to a final volume of 170 Ml/g of the originaltissue weight. The binding assay consisted of 1 Ml in 12×75 mmpolypropylene tubes: 50 uL ³H-bradykinin (20,000 dpm, ˜0.3 Nm in thefinal assay volume), 100 L displacing drug in buffer A, and 750 Ultissue homogenate. Each tray contained tubes, to which no drug was addedto measure maximum binding and tubes to which bradykinin (1 uM finalconcentration) had been added, to measure specific binding. Specificbinding accounted for 96-98% total binding. Tubes were incubated for 90min at ambient temperature. The assays were terminated by filtrationover Whatman GF/B glass fiber filters that had been pretreated for 2hours with polyethyleneimine (2 g/L) using a Brandel Tissue Harvester,followed by washing with 4×1 Ml aliquots of ice-cold 50 Mm Tris, Ph 7.4.Filters were dissolved in Ready-Safe Fluor (Beckman) for at least 90 minbefore quantitation by liquid scintillation spectrometry. Kd values weredetermined using saturation binding and analysis by EBDA [(G. A.MacPherson, J. Pharmacol. Methods, (1985), 213)], followed by LIGAND [P.J. Munson, D. Rodbard, Anal. Biochem., (1980), 220]. Ki values weredetermined using competitive analysis followed by EBDA and LIGAND. Thefollowing test results were obtained.

Test Compound Ki (mM)  1) D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 11.54(Hydroxyproline trans phenyl carbamoyl)-Arg  2)D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 1.83 (Hydroxyproline cis methylether)-Arg  3) D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 1.58(Hydroxyproline cis propyl ether)-Arg  4)D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 4.85 (Hydroxyproline trans propylether)-Arg  5) D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 3.76(Hydroxyproline cis 2-nitrophenyl +\−0.89 ether)-Arg  6)D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 3.98 (Hydroxyproline cis4-nitrophenyl +\−1.5 ether)-Arg  7)D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 4.15 (Hydroxyproline cis ethylether)-Arg +\−0.58  8) D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 13.25(Hydroxyproline cis cyclohexyl-methyl +\−1.55 ether)-Arg  9)D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 0.06 (Hydroxyproline cis phenylthioether)-Arg +\−0.01 10) D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 2.51(Hydroxyproline cis phenyl ether)-Arg +\−0.06 11)D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 5.44 (hydroxyproline trans ethylether)-Arg

Determination of Bradykinin Antagonist Activity

This protocol was designed to identify compounds that possess antagonistactivity at bradykinin receptors on intestinal (ileal longitudinal)smooth muscle.

Guinea pig intestine was removed and placed in a Petri dish containingTyrodes solution and cut into 3-4 cm segments. The longitudinal musclewas separated from the underlying circular muscle using a cottonapplicator (Paton and Zar, J. Physiol. (1968), 194:13). Muscle stripswere connected to isometric force-displacement transducers (Grass orGould) coupled to a physiograph and placed in tissue baths containingTyrode's solution at 37° C. Each preparation was suspended under aresting tension of 2 g.

After equilibration of the tissues, appropriate volumes of bradykininsolutions were cumulatively added to the 10 Ml tissue baths to increasethe concentration of bradykinin in the bath step-by-step without washingout after each single dose. Higher concentrations were added only afterthe preceding contraction had reached a steady value. When the nextconcentration step does not cause a further increase in contraction, itwas assumed that the maximum effect had been obtained and the tissue waswashed to remove bradykinin and allowed to recover for 15 minutes.Antagonism of bradykinin responses in the presence of antagonist weredetermined by repeating the cumulative addition procedure for bradykininafter the tissue has been exposed to the antagonist for 5 minutes. Threeor four differentconcentrations of antagonist are studied sequentiallyin the same preparations. Responses were expressed as a percentage ofthe maximum contraction elicited by bradykinin in the absence ofantagonist. pA2 values were calculated by Schild analysis. The followingresults were obtained.

Test Compound pA₂ 1) D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 6.85(Hydroxyproline cis methyl ether)-Arg 2)D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 6.65 (Hydroxyproline cis propylether)-Arg 3) D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic- 6.86 (Hydroxyprolinetrans propyl ether)-Arg 4) D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 6.85(Hydroxyproline cis 2-nitrophenyl ether)-Arg 5)D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 6.86 (Hydroxyproline cis4-nitrophenyl +\−0.02 ether) -Arg 6)D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 7.29 (Hydroxyproline trans ethylether)- Arg 7) D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 7.95(Hydroxyproline-cis phenylthioether)- Arg 8)D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic- 6.96 (Hydroxyproline-cis ethylether)- Arg

What is claimed is:
 1. A peptide having the formula:N-A-B-C-D-E-F-G-H-I-J-Cn, wherein N is hydrogen; A is D-Arg; B is Arg; Cand D are independently selected from the group consisting of Pro and⁴Hyp; E is Gly; F is selected from the group consisting of Phe, Leu, andThi; G is a direct bond or is Ser; H is a compound of theD-configuration selected from the consisting of D-Phe and D-Tic; I isselected from the group consisting of Aoc and compounds of the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, isobutyl, cyclohexylmethyl, allyl, methallyl, prenyl, benzyl,phenyl, nitrophenyl, phenylpropyl, and methylbutyl, and wherein X iseither sulfur or oxygen; J is Arg; Cn is a hydroxyl group; andpharmaceutically acceptable salts thereof.
 2. A peptide having theformula: N-A-B-C-D-E-F-G-H-I-J-Cn wherein N is hydrogen; A is selectedfrom the group consisting of L-Arg, D-Arg, Lys-Lys, and Lys; B isselected from the group consisting of L-Arg, D-Arg, and Lys; C and D areindependently selected from the group consisting of Pro, dehydroPro, and4Hyp; E is Gly; F is selected from the group consisting of Phe, Leu andThi; G is direct bond or is selected from the group consisting of Serand Thr; H is selected from the group consisting of D-Phe and D-Tic; Iis a compound having the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, isobutyl, cyclohexylmethyl, allyl, prenyl, methallyl, benzyl,phenyl, nitrophenyl, phenylpropyl, and methylbutyl, and wherein X issulfur or oxygen; J is Arg; Cn is a hydroxyl group; and pharmaceuticallyacceptable salts thereof.
 3. A peptide of claim 1 wherein: A is D-Arg; Bis Arg; C and D are selected from the group consisting of Pro and 4Hyp;E is Gly; F is Phe; G is Ser; H is D-Phe; I is a compound having theformula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, and phenyl and X is sulfur or oxygen; J is Arg; andpharmaceutically acceptable salts thereof.
 4. A peptide of claim 1wherein: A is D-Arg; B is Arg; C and D are selected from the groupconsisting of Pro and 4Hyp; E is Gly; F is Phe; G is Ser; H is D-Tic; Iis a compound having the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, phenyl and nitrophenyl and X is sulfur or oxygen; J is Arg; andpharmaceutically acceptable salts thereof.
 5. A peptide of claim 1wherein: A is D-Arg; B is Arg; C and D are selected from the groupconsisting of Pro and 4Hyp; E is Gly; F is Phe; G is a direct bond; H isselected from the group consisting of D-Phe and D-Tic; I is a compoundhaving the formula:

wherein R is selected from the group consisting of methyl, ethyl,propyl, phenyl and nitrophenyl and X is sulfur or oxygen; J is Arg; andpharmaceutically acceptable salts thereof.
 6. A peptide of claim 1wherein: A is D-Arg; B is Arg; C and D are selected from the groupconsisting of Pro and 4Hyp; E is Gly; F is Phe; G is Ser; H is D-Tic; Iis a compound having the formula:

wherein R is methyl and X is oxygen; J is Arg; and pharmaceuticallyacceptable salts thereof.
 7. A peptide of claim 1 wherein: A is D-Arg; Bis Arg; C and D are selected from the group consisting of Pro and 4Hyp;E is Gly; F is Phe; G is Ser; H is D-Tic; I is a compound having theformula:

wherein R is ethyl and X is oxygen; J is Arg; and pharmaceuticallyacceptable salts thereof.
 8. A peptide of claim 1 wherein: A is D-Arg; Bis Arg; C and D are selected from the group consisting of Pro and 4Hyp;E is Gly; F is Phe; G is Ser; H is D-Tic; I is a compound having theformula:

wherein R is propyl and X is oxygen; J is Arg; and pharmaceuticallyacceptable salts thereof.
 9. A peptide of claim 1 wherein: A is D-Arg; Bis Arg; C and D are selected from the group consisting of Pro and 4Hyp;E is Gly; F is Phe; G is Ser; H is D-Tic; I is a compound having theformula:

wherein R is phenyl and X is oxygen; J is Arg; and pharmaceuticallyacceptable salts thereof.
 10. A peptide of claim 1 wherein: A is D-Arg;B is Arg; C and D are selected from the group consisting of Pro and4Hyp; E is Gly; F is Phe; G is Ser; H is D-Tic; I is a compound havingthe formula:

wherein R is allyl and X is oxygen; J is Arg; and pharmaceuticallyacceptable salts thereof.
 11. A peptide of claim 1 wherein: A is D-Arg;B is Arg; C and D are selected from the group consisting of Pro and4Hyp; E is Gly; F is Phe; G is Ser; H is D-Tic; I is a compound havingthe formula:

wherein R is methyl and X is sulfur; J is Arg; and pharmaceuticallyacceptable salts thereof.
 12. A peptide of claim 1 wherein: A is D-Arg;B is Arg; C and D are selected from the group consisting of Pro and4Hyp; E is Gly; F is Phe; G is Ser; H is D-Tic; I is a compound havingthe formula:

wherein R is ethyl and X is sulfur; J is Arg; and pharmaceuticallyacceptable salts thereof.
 13. A peptide of claim 1 wherein: A is D-Arg;B is Arg; C and D are selected from the group consisting of Pro and4Hyp; E is Gly; F is Phe; G is Ser; H is D-Tic; I is a compound havingthe formula:

wherein R is propyl and X is sulfur; J is Arg; and pharmaceuticallyacceptable salts thereof.
 14. A compound selected from the groupconsisting of: D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyprolinetrans methyl ether)-Arg,D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline trans propylether)-Arg, D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline cispropyl ether)-Arg, D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyprolinecis methyl ether)-Arg,D-Arg-Arg-Pro-4Hyp-Gly-Thi-Ser-D-Tic-(Hydroxyproline transphenylcarbamoyl)-Arg,D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis 2-nitrophenylether)-Arg, D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis4-nitrophenyl ether)-Arg,D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis ethylether)-Arg, D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline ciscyclohexylmethyl ether)-Arg,D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline cis phenylthioether)-Arg, and D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyprolinecis phenyl ether)-Arg,D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline trans ethylether)-Arg D-Arg-Arg-Pro-4Hyp-Gly-Phe-Ser-D-Tic-(Hydroxyproline transphenyl thioether)-Arg.
 15. A pharmaceutical composition comprising apharmaceutical carrier and an effective amount of the peptide of claim 1to antagonize bradykinin receptor activity.
 16. A pharmaceuticalcomposition comprising a pharmaceutical carrier and an effective amountof the peptide of claim 3 to antagonize bradykinin receptor activity.17. A pharmaceutical composition comprising a pharmaceutical carrier andan effective amount of the peptide of claim 14 to antagonize bradykininreceptor activity.